Project Summary Benzodiazepines (BZs) are therapeutic drugs widely used to treat anxiety, insomnia and seizure disorders and as additional drug therapy in schizophrenia and depression. BZs bind and potentiate the inhibitory activity of specific subtypes of GABA type A receptors (GABAARs). Despite the efficacy of BZs, their prolonged use is severely limited by tolerance, dependence and withdrawal. Very little is mechanistically known about the neuroadaptations that underlie a BZ tolerant brain state. Chronic BZ treatment results in a decrease in BZ potentiation of GABA activity at GABAARs, suggesting changes in receptor subunit composition and/or function at inhibitory synapses. Furthermore, the neuroplasticity occurring during BZ treatment is also dependent on excitatory glutamatergic N-methyl-D-aspartate receptors (NMDAR), as co- administration of NMDAR antagonists can prevent BZ tolerance. Using a high throughput quantitative proteomic approach to investigate BZ sedative tolerance-induced changes in the rodent cortex our data show for the first time significant upregulation of key excitatory synapse components. Together with our prior work showing that sustained BZ exposure decreases synaptic levels of BZ sensitive GABAARs, these findings generated the central hypothesis: Benzodiazepine treatment reduces benzo- sensitive GABAAR subtype signaling concomitant with enhancing excitatory synapse strength. The proposed research capitalizes on the use of novel optical methods and quantitative proteomics to address the critical knowledge gap in how prolonged BZ use induces neuroplasticity in both inhibitory GABAAR and excitatory NMDAR signaling. Two independent and complementary Aims are proposed to test these mechanistic components of BZ tolerance. The first aim will use quantitative mass spectrometry, electrophysiology, behavioral and pharmacological methods to define and functionally assess in vivo BZ treatment induced changes in both inhibitory and excitatory synapses of the rodent cortex. The second aim will apply high resolution imaging techniques and an innovative optical biosensor for BZ sensitive GABAAR combined with genetic, biochemical and electrophysiological approaches to identify BZ treatment induced GABAAR post translational modifications and cellular mechanisms leading to rapid BZ uncoupling and the progression to BZ tolerance. In vitro cellular and neuronal studies will be complimented by evaluation of GABAAR post-translational modifications as BZ sedative tolerance develops in mice. These findings will provide new directions for the development of therapeutic approaches to mitigate or avoid BZ tolerance, addressing a significant unmet public health need.